- Email: [email protected]
COMBINATORIAL TREATMENT WITH AB IMMUNOTHERAPY AND BDNF IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
P1038
Poster Presentations: Wednesday, July 27, 2016
terminal 31-aa peptide C31. We previously found that if we block this C-terminal cleavage of APP in vivo by mutating the cleavage site, many mouse AD phenotypes were ameliorated. This work describes our progress toward discovering small molecule inhibitors of this destructive process. Methods: Using the D664 (APP695) cleavage site-specific antibody (APP delta C31, Enzo), we measured the level of N-terminal fragment resulting from this intracellular APP proteolysis. We developed an AlphaLISA assay for library screening purposes and a commercial ELISA for validation and research purposes for this C-terminal cleavage. For our library screening, the cell model system used was CHO cells stably transfected with APP770 (CHO-7W). We stimulated cleavage at the D739 caspase cleavage site (APP770) by treating these cells with cerevastatin and were able to reduce this cleavage and rescue cell death with the pan-caspase inhibitor Q-VD-OPh. An ex vivo I5 (hAPPwt) mouse hippocampal slice culture model was also utilized to validate active compounds. Results: Several classes and individual compounds were found to be effective at reducing C-terminal APP caspase cleavage and rescuing cell death in a dose-dependent manner. The most effective include SERCA inhibitors, inhibitors of Wnt signaling and calcium channel antagonists. Conclusions: We have identified and validated individual and classes of compounds that inhibit C-terminal APP caspase cleavage and rescue cell death in a dose–dependent manner. The resulting N-terminal APP fragment could be an important AD biomarker and blocking this cleavage could prove to be an important AD therapeutic approach. P4-063
COMBINATORIAL TREATMENT WITH AB IMMUNOTHERAPY AND BDNF IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Fernando Calvo Baltanas1, Alan H. Nagahara1, Jazmin B. Florio1, Michael Mante1, Edward Rockenstein1, Charles J. Heyser1, Eliezer Masliah2, Mark H. Tuszynski1, 1University of California San Diego, La Jolla, CA, USA; 2University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Background: Both amyloid immunotherapy, and brain-derived neurotrophic factor (BDNF) gene therapy to the entorhinal cortex, exhibit beneficial effects in animal models of AD. Combination therapy may present the best opportunity to detect clinical benefit in human clinical trials. To address this possibility, we have designed a study to combine Ab immunotherapy with BDNF gene delivery to the entorhinal cortex in APP transgenic mice. When adeno-associated virus serotype 2 (AAV2) is injected into the entorhinal cortices of mice, rats or non-human primates, BDNF levels increase throughout the entorhinal cortex and hippocampus, targeting key neural circuitry involved in early AD. Ab immunotherapy targets a key pathogenic mechanism in AD, while BDNF gene therapy can directly prevent neuronal death and stimulate neuronal function: these two approaches target distinct mechanisms in AD models, potentially offering additive or synergistic benefits. Methods: At age 6 mo, APP transgenic male mice (Line 41 strain; Swedish KM670/671NL and London V717I mutations) or wildtype controls receive active immunization with Ab peptide (human Ab1-42; 100 mg/injection) and complete Freund’s adjuvant, with monthly injections of Aß peptide for 5 more months; additional APP mice or wild-type mice received PBS. At 11 mo of age, mice received bilateral infusions of AAV2-BDNF or AAV2-GFP vectors (1x109vg/ml, 10 ml total) into the entorhinal cortices. One month later, 4 APP-tg groups (non-treated, Ab immunized, BDNF-treated, and Ab immunized/BDNF-treated) and wild-type mice are tested on hippocampal-dependent memory tasks. This
portion of studies has been completed and we are currently examining brains for synaptic measures, functional cell markers, Ab levels and additional labels. Results: A comprehensive report of the findings will be presented to determine whether combinatorial treatment with AAV2-BDNF and Ab immunization exhibit greater neuroprotective or neurorestorative properties in this mouse model of AD. Conclusions: Findings of this study will inform the decision process for proceeding to translational testing of combinatorial therapy with BDNF and anti-amyloid therapies for AD. P4-064
HUMAN AMYLOID BETA EXPRESSED IN DROSOPHILA: OLIGOMERIZATION AND TOXICITY
Maria Luisa Moro, Delphine Boche, Amrit Mudher, University of Southampton, Southampton, United Kingdom. Contact e-mail: [email protected] Background: In Alzheimer’s disease, cognitive impairment has
shown poor correlation with the number of plaques composed by amyloid-beta (A-beta). This has enforced the hypothesis that small A-beta assemblies, like oligomers, may be instead the species mediating A-beta neurotoxicity and drive the cognitive decline. According to the stage of aggregation, A-beta oligomers can have a different level of solubility in denaturing solvents. We decided to validate, in an invertebrate organism, the hypothesis that the toxicity of Abeta is linked to its oligomerization, expressing human wild type A-beta1-42 in Drosophila Melanogaster. Methods: We employed fly stocks where A-beta1-42 was cloned into the Gal4responsive pUAST expression vector and the transgene inserted on chromosome 2 (single transgenic Abeta flies) or on chromosome 2 and 3 (double transgenic Abeta flies). The A-beta peptide was expressed by crossing transgenic flies with the Gal4-elavc155 panneuronal driver strain. To detect A-beta monomers and oligomers, fly heads were homogenized in 2% SDS buffer and the soluble and insoluble fraction analysed by Western Blot, using Abeta specific antibodies. Viable transgenic and control (Gal4-elavc155) flies were counted every two days to assess their longevity. Results: In the double transgenic flies, we detected A-beta monomer and dimers in the SDS soluble fraction and tetramers in the SDS insoluble fraction of the head homogenate. Differently, in the single transgenic flies, we detected the monomer only in the SDS soluble fraction and no A-beta species in the SDS insoluble fraction. The highest lifespan reduction, compared to control flies, was found in the double transgenic flies in association with detectable Abeta oligomers. Conclusions: The analysis of these two transgenic models confirms that A-beta oligomerization is critical for A-beta toxicity and that invertebrates, like Drosophila, can be used to assess in vivo biological effects of A-beta aggregation. P4-065
IMPACT OF APOE POLYMORPHISMS ON AMYLOID-BETA OLIGOMERIZATION AND ASSOCIATED COGNITIVE DECLINE
Nicholas F. Fitz, Alexis Y. Carter, Kyong Nyon Nam, Emilie L. Castranio, Anais Mounier, Cody M. Wolfe, Iliya Lefterov, Radosveta Koldamova, University of Pittsburgh, Pittsburgh, PA, USA. Contact e-mail: nffitz@pitt. edu Background: Although the inheritance of APOE4 allele of APOE is
the major genetic risk factor for late-onset AD, the mechanisms underlying this association remain elusive. APOE can differentially modulate Ab accumulation and clearance through blood brain barrier (BBB), or Ab degradation by astrocytes and microglia. There are two conflicting views of the effect of APOE4 on AD pathology:
Poster Presentations: Wednesday, July 27, 2016
terminal 31-aa peptide C31. We previously found that if we block this C-terminal cleavage of APP in vivo by mutating the cleavage site, many mouse AD phenotypes were ameliorated. This work describes our progress toward discovering small molecule inhibitors of this destructive process. Methods: Using the D664 (APP695) cleavage site-specific antibody (APP delta C31, Enzo), we measured the level of N-terminal fragment resulting from this intracellular APP proteolysis. We developed an AlphaLISA assay for library screening purposes and a commercial ELISA for validation and research purposes for this C-terminal cleavage. For our library screening, the cell model system used was CHO cells stably transfected with APP770 (CHO-7W). We stimulated cleavage at the D739 caspase cleavage site (APP770) by treating these cells with cerevastatin and were able to reduce this cleavage and rescue cell death with the pan-caspase inhibitor Q-VD-OPh. An ex vivo I5 (hAPPwt) mouse hippocampal slice culture model was also utilized to validate active compounds. Results: Several classes and individual compounds were found to be effective at reducing C-terminal APP caspase cleavage and rescuing cell death in a dose-dependent manner. The most effective include SERCA inhibitors, inhibitors of Wnt signaling and calcium channel antagonists. Conclusions: We have identified and validated individual and classes of compounds that inhibit C-terminal APP caspase cleavage and rescue cell death in a dose–dependent manner. The resulting N-terminal APP fragment could be an important AD biomarker and blocking this cleavage could prove to be an important AD therapeutic approach. P4-063
COMBINATORIAL TREATMENT WITH AB IMMUNOTHERAPY AND BDNF IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Fernando Calvo Baltanas1, Alan H. Nagahara1, Jazmin B. Florio1, Michael Mante1, Edward Rockenstein1, Charles J. Heyser1, Eliezer Masliah2, Mark H. Tuszynski1, 1University of California San Diego, La Jolla, CA, USA; 2University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Background: Both amyloid immunotherapy, and brain-derived neurotrophic factor (BDNF) gene therapy to the entorhinal cortex, exhibit beneficial effects in animal models of AD. Combination therapy may present the best opportunity to detect clinical benefit in human clinical trials. To address this possibility, we have designed a study to combine Ab immunotherapy with BDNF gene delivery to the entorhinal cortex in APP transgenic mice. When adeno-associated virus serotype 2 (AAV2) is injected into the entorhinal cortices of mice, rats or non-human primates, BDNF levels increase throughout the entorhinal cortex and hippocampus, targeting key neural circuitry involved in early AD. Ab immunotherapy targets a key pathogenic mechanism in AD, while BDNF gene therapy can directly prevent neuronal death and stimulate neuronal function: these two approaches target distinct mechanisms in AD models, potentially offering additive or synergistic benefits. Methods: At age 6 mo, APP transgenic male mice (Line 41 strain; Swedish KM670/671NL and London V717I mutations) or wildtype controls receive active immunization with Ab peptide (human Ab1-42; 100 mg/injection) and complete Freund’s adjuvant, with monthly injections of Aß peptide for 5 more months; additional APP mice or wild-type mice received PBS. At 11 mo of age, mice received bilateral infusions of AAV2-BDNF or AAV2-GFP vectors (1x109vg/ml, 10 ml total) into the entorhinal cortices. One month later, 4 APP-tg groups (non-treated, Ab immunized, BDNF-treated, and Ab immunized/BDNF-treated) and wild-type mice are tested on hippocampal-dependent memory tasks. This
portion of studies has been completed and we are currently examining brains for synaptic measures, functional cell markers, Ab levels and additional labels. Results: A comprehensive report of the findings will be presented to determine whether combinatorial treatment with AAV2-BDNF and Ab immunization exhibit greater neuroprotective or neurorestorative properties in this mouse model of AD. Conclusions: Findings of this study will inform the decision process for proceeding to translational testing of combinatorial therapy with BDNF and anti-amyloid therapies for AD. P4-064
HUMAN AMYLOID BETA EXPRESSED IN DROSOPHILA: OLIGOMERIZATION AND TOXICITY
Maria Luisa Moro, Delphine Boche, Amrit Mudher, University of Southampton, Southampton, United Kingdom. Contact e-mail: [email protected] Background: In Alzheimer’s disease, cognitive impairment has
shown poor correlation with the number of plaques composed by amyloid-beta (A-beta). This has enforced the hypothesis that small A-beta assemblies, like oligomers, may be instead the species mediating A-beta neurotoxicity and drive the cognitive decline. According to the stage of aggregation, A-beta oligomers can have a different level of solubility in denaturing solvents. We decided to validate, in an invertebrate organism, the hypothesis that the toxicity of Abeta is linked to its oligomerization, expressing human wild type A-beta1-42 in Drosophila Melanogaster. Methods: We employed fly stocks where A-beta1-42 was cloned into the Gal4responsive pUAST expression vector and the transgene inserted on chromosome 2 (single transgenic Abeta flies) or on chromosome 2 and 3 (double transgenic Abeta flies). The A-beta peptide was expressed by crossing transgenic flies with the Gal4-elavc155 panneuronal driver strain. To detect A-beta monomers and oligomers, fly heads were homogenized in 2% SDS buffer and the soluble and insoluble fraction analysed by Western Blot, using Abeta specific antibodies. Viable transgenic and control (Gal4-elavc155) flies were counted every two days to assess their longevity. Results: In the double transgenic flies, we detected A-beta monomer and dimers in the SDS soluble fraction and tetramers in the SDS insoluble fraction of the head homogenate. Differently, in the single transgenic flies, we detected the monomer only in the SDS soluble fraction and no A-beta species in the SDS insoluble fraction. The highest lifespan reduction, compared to control flies, was found in the double transgenic flies in association with detectable Abeta oligomers. Conclusions: The analysis of these two transgenic models confirms that A-beta oligomerization is critical for A-beta toxicity and that invertebrates, like Drosophila, can be used to assess in vivo biological effects of A-beta aggregation. P4-065
IMPACT OF APOE POLYMORPHISMS ON AMYLOID-BETA OLIGOMERIZATION AND ASSOCIATED COGNITIVE DECLINE
Nicholas F. Fitz, Alexis Y. Carter, Kyong Nyon Nam, Emilie L. Castranio, Anais Mounier, Cody M. Wolfe, Iliya Lefterov, Radosveta Koldamova, University of Pittsburgh, Pittsburgh, PA, USA. Contact e-mail: nffitz@pitt. edu Background: Although the inheritance of APOE4 allele of APOE is
the major genetic risk factor for late-onset AD, the mechanisms underlying this association remain elusive. APOE can differentially modulate Ab accumulation and clearance through blood brain barrier (BBB), or Ab degradation by astrocytes and microglia. There are two conflicting views of the effect of APOE4 on AD pathology: